The many different cell types found in blood are all derived from pluripotent hematopoietic stem cells. Stem cells perform two functions: (1) they reproduce themselves, thereby maintaining a stem cell population in the body and (2) they provide progeny cells committed to differentiate into any of the mature blood cell types. The cell which is committed to differentiate along a particular hematopoietic pathway is termed a progenitor cell. Progenitor cells for T lymphocytes, B lymphocytes, granulocytes, red blood cells, platelets, and eosinophils, as well as earlier progenitors which can individually give rise to several of the mature cell types, have been studied experimentally both in vivo and in vitro (Dexter, T. M. 1983 J. Pathology 141 415-433). It has been determined in vitro that proliferation and/or differentiation of each progenitor cell type depends upon specific "factors" which have been derived from various sources. For example, the later progenitors of red blood cells require a factor called erythropoietin. The factors required for survival, proliferation and differentiation of the myeloid progenitors committed to form mature neutrophilic granulocytes, monocytes and mature macrophages are called colony stimulating factors (CSFs).
CSF activity has been studied extensively in the mouse. Most adult mouse organs produce CSF activity. However, compositions containing CSF activity that have been obtained from various tissues and by various methods appear to differ in their biochemical characteristics. Thus, the structural relationships between the different factors remain unknown. Furthermore, CSF activity appears to act at more than one step of granulocyte and macrophage development, and again it has been uncertain whether a single factor is responsible for all of the observed activities or whether a different factor acts at each step. (Burgess, A. and Metcalf, D. 1980 Blood 56 947-957).
Human CSF active has been obtained from placenta, certain fetal tissues, macrophages, and stimulated T cells. A line of T cells (Mo) that produces one or more potent CSF activities was established from a patient with a T cell variant of hairy cell leukaemia (leukaemic reticuloendotheliosis) (Golde et al 1978 Blood 52 1068-1072).
The ability of CS activity to stimulate granulocyte and macrophage production indicated that pharmaceutical compositions having CSF activity are clinically useful in situations where increased production of these (myeloid) cell types is required. Indeed, several patients with extremely high levels of apparently normal circulating granulocytes have been shown to have tumors which over-produce CSF. In one case, upon surgical removal of the tumor, the granulocyte count rapidly declined towards a normal level, strongly suggesting that CSF may be useful in regulating the numbers of circulating granulocytes. (Hocking, W., Goodman, J., and Golde, D. Blood 61 600 (1983)). In particular, CSF compositions are useful clinically for the treatment of myelo-suppression caused by chemotherapeutical or irradiation treatment of cancer. In addition, CSF compositions are useful in treating severe infections because CSF can increase and/or activate the number of granulocytes and/or monocytes.
There are various different types of known CSF activities, including granulocyte. CSF (G-CSF), macrophage-CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF) and multi-CSF. The present invention is particularly concerned with GM-CSF. CSF proteins are known from various animal sources. However, the present invention is particularly concerned with primate CSF, more particularly human CSF and ape CSF.
Biological and biochemical characterization of compositions having CSF activity, and study of these compositions in the clinical setting have been hampered to date by the scarcity and impurity of human and/or other primate CSF compositions. It can be appreciated that it would be desirable to identify the protein or proteins responsible for CSF activity. Furthermore, it would be desirable to have a primate, preferably human source of such CSF that could readily supply these proteins in quantities and purity sufficient for biological and biochemical characterization and for use as therapeutic agents.
Recently developed techniques of molecular cloning make it possible to clone a nucleotide sequence which encodes a protein and to produce that protein in quantity using a suitable host-vector system Maniatis, T. Molecular Cloning--A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1982). The protein can then be recovered by known separation and purification techniques. Cloning methods which have been used to date can be grouped into three general categories: (1) methods based upon knowledge of the protein structure, for example, its amino acid sequence; (2) methods based upon identification of the protein expressed by the cloned gene using an antibody specific for that protein; and (3) methods based upon identification of an RNA species which can be translated to yield the protein or activity encoded by the gene of interest.
Each of these classes of methods becomes difficult to apply when the protein of interest, such as CSF protein, is available in very low amount. Thus, if it is difficult to obtain an adequate quantity of purified protein, then it is difficult to determine the amino acid sequence or even partial sequences of the protein. Similarly, identification of an expressed protein by antibody binding is preferentially carried out using a high-titer monospecific polyclonal antiserum. Such an antiserum cannot be obtained in the absence of quantities of the pure protein (antigen). A monoclonal antibody offers an alternative approach, but the required antibody can also be difficult to obtain in the absence of suitable antigen, and such monoclonal antibody may not react with the protein in the form in which the protein is expressed by available recombinant host-vector systems. Finally, translation of an RNA species to yield an identifiable protein or activity requires that the RNA in question be present in the RNA source in sufficient abundance to give a reliable protein or activity signal. The relative abundance of an RNA encoding a particular protein generally parallels the abundance of the protein, so that a rare protein is usually encoded by a rare mRNA.
The Mo cell line has been used both as a starting material for purifying human CSFs and for identifying the corresponding messenger RNAs. However, even with this relatively good source of CSF activity, it has proved to be extremely difficult to isolate enough of the protein for structural studies.
In order to overcome the problems inherent in cloning the nucleotide sequence encoding a rare protein such as CSF by the methods described above, a novel method was developed. This method requires only that the gene product or its activity can be reliably measured. Suitable methods of CSF assay are described in Example 2 hereinafter. In a second aspect, a purification process has been developed which enables the CSF protein to be isolated and purified from either recombinant or natural sources in a level of purity and activity much higher than was previously possible.
Summary of the recombinant DNA process of the Invention
In its first aspect the present invention overcomes the problems of the prior art and provides a ready source of protein having CSF activity using recombinant DNA technology. In accord with the present invention, a novel cloning technique that requires only an assay for CSF activity is utilized to clone cDNA coding for a protein having CSF activity. Thus, the present invention provides a cDNA coding for a protein having CSF activity (i.e. CSF/cDNA), a microorganism or cell line transformed with a recombinant vector containing such CSF/cDNA, and a method for producing CSF protein by expressing said CSF/cDNA by culturing a microorganism or cell line. Because the CSF protein is produced from a clone in accord with the present invention, we can be sure that it is a protein that has CSF activity. The invention further comprises a method for preparing and isolating a transformation vector containing CSF/cDNA, said method comprising:
preparing RNA from a cell that produces CSF; PA1 preparing polyadenylated messenger RNA from said RNA; PA1 preparing single stranded cDNA from said messenger RNA; PA1 converting the single stranded cDNA to double stranded cDNA; PA1 inserting the double stranded cDNA into transformation vectors and transforming bacteria with said vector to form colonies; PA1 picking pools of 200 to 500 colonies each and isolating plasmid DNA from each pool; PA1 transfecting the plasmid DNA into suitable host cells for expressing CSF protein; PA1 culturing the transfected cells and assaying the supernatant for CSF activity; and PA1 selecting CSF positive pools and screening the colonies used to make the pool to identify a colony having CSF activity.
The CSF proteins of this invention are growth and differentiation hormones for the cells of the myeloid system. They are for example indicated for use clinically for the treatment of myelo-suppression especially (symptomatic) granulocyto-penia following chemotherapeutical or irradiation treatment of cancer.